Myocardial ischemia negatively impacts cardiac function through: 1) Immediate post-ischemic contractile dysfunction of non-infarcted, reperfused myocardium (stunning);2) Myocardial infarction and infarct extension;and 3) Delayed left ventricular remodeling. Since cardiac ischemia and infarction are associated with increased free cytosolic calcium, activation of calpains (calcium-activated cystein proteases) is thought to contribute to ischemic cardiac injury. Limb-Girdle Muscular Dystrophy 2A (or "calpainopathy") in patients with loss of function mutations in the calpain-3 gene result reveals an essential function for calpains in skeletal muscle health. We have created a myocardial calpain loss-of-function mouse (transgenic overexpression of calpain-inhibitory calpastatin) which develops a cardiomyopathy with many of the histologic features of skeletal calpainopathy. Based on these observations, our GENERAL HYPOTHESIS is that calpains 1 and 2 have unique functions in normal cardiac health, and due to differential activation by calcium and differential mRNA regulation after infarction, play distinct roles in mediating post-ischemic injury. To test this hypothesis we created cardiac-specific calpain 1 and 2 knockout and conditional overexpression mice, and will pursue these SPECIFIC AIMS: SA #1 -Define the normal functions of myocardial calpain 1 and calpain 2 in studies of cardiac structure, function, and cell signaling. SA#2-Delineate the pathophysiological consequences and relevant cellular mechanisms of calpain 1 and calpain 2 activation after myocardial infarction and ischemia-reperfusion injury by defining the relative roles of apoptotic and necrotic cardiomyocyte death. SA#3-Determine the biochemical mechanisms for calpain-mediated caspase activation and stimulation of apoptosis in studies of calpain 1 and 2-modulated cultured neonatal and adult cardiac myocytes. Collectively, these studies will apply state-of-the-art techniques for in vivo genetic manipulation, microphysiologic analysis, and biochemical assessment to achieve insight into the roles of myocardial calpains in health and disease.